Function of astrocyte MyD88 in high-fat-diet-induced hypothalamic inflammation

Background A growing body of evidence shows that hypothalamic inflammation is an important factor in the initiation of obesity. In particular, reactive gliosis accompanied by inflammatory responses in the hypothalamus are pivotal cellular events that elicit metabolic abnormalities. In this study, we examined whether MyD88 signaling in hypothalamic astrocytes controls reactive gliosis and inflammatory responses, thereby contributing to the pathogenesis of obesity. Methods To analyze the role of astrocyte MyD88 in obesity pathogenesis, we used astrocyte-specific Myd88 knockout (KO) mice fed a high-fat diet (HFD) for 16 weeks or injected with saturated free fatty acids. Astrocyte-specific gene expression in the hypothalamus was determined using real-time PCR with mRNA purified by the Ribo-Tag system. Immunohistochemistry was used to detect the expression of glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, phosphorylated signal transducer and activator of transcription 3, and α-melanocyte-stimulating hormone in the hypothalamus. Animals’ energy expenditure was measured using an indirect calorimetry system. Results The astrocyte-specific Myd88 KO mice displayed ameliorated hypothalamic reactive gliosis and inflammation induced by injections of saturated free fatty acids and a long-term HFD. Accordingly, the KO mice were resistant to long-term HFD-induced obesity and showed an improvement in HFD-induced leptin resistance. Conclusions These results suggest that MyD88 in hypothalamic astrocytes is a critical molecular unit for obesity pathogenesis that acts by mediating HFD signals for reactive gliosis and inflammation.


Background
The increasing rate of obesity in the global population has become a major public health problem. Therefore, many investigations have been performed to identify the underlying mechanisms and pathological components of obesity [1,2]. In particular, it has been proposed that perturbation of the hypothalamic neuronal circuit that controls whole-body energy metabolism is a primary cause of obesity development [3][4][5]. During the past decade, a great deal of attention has been paid to investigating the hypothalamic neuronal circuit linked to whole-body energy metabolism under the control of afferent inputs derived from metabolically involved peripheral organs [6][7][8].
Astrocytes are the most abundant cells in the central nervous system and dynamically participate in maintaining normal neuronal functions by playing multiple supportive roles. Thus, a growing body of evidence has emerged linking metabolic processes in hypothalamic astrocytes with the physiological or pathological control of body energy balance [9][10][11]. According to the recent literature, neuroinflammation and reactive gliosis can be observed in the hypothalami of mice exposed to a highfat diet (HFD) before the occurrence of significant body weight gain, and this is sustained with continuous HFD feeding, suggesting that hypothalamic gliosis accompanied by inflammation is a crucial cellular event in obesity pathogenesis [12][13][14][15]. Thus, unmasking the underlying mechanism by which an HFD induces hypothalamic inflammation and gliosis is required to better understand the initiation and deterioration of metabolic disorders caused by over-nutrition.
Myeloid differentiation primary response 88 (MyD88) is a crucial adaptor molecule of Toll-like receptor (TLR) signaling that initiates innate immunity by mediating a variety of humoral factors and infectious pathogens [16][17][18]. In particular, the TLR and MyD88 axis in the hypothalamus is a key player in HFD-induced hypothalamic inflammatory responses. Mice fed with an HFD expressed high levels of TLR4 and MyD88 in the hypothalamus, which was coupled with intracellular inflammatory signaling cascades such as the Jun kinase and nuclear factor kappa B (NF-κB) pathways [19,20]. A recent study reported that interaction between circulating saturated free fatty acids (sFFAs) and TLR4 was involved in the hypothalamic control of energy homeostasis and that mice bearing neuronspecific deletion of the Myd88 gene were protected against HFD-induced obesity through the alleviation of hypothalamic inflammation and leptin resistance [21]. However, it is unclear whether TLR and MyD88 signaling in astrocytes is triggered by over-nutrition and thus directly linked to the development of obesity in association with hypothalamic gliosis and inflammation. In this study, we investigated whether MyD88 signaling in astrocytes is involved in hypothalamic inflammation and reactive gliosis and whether altering the activity of MyD88 signaling in astrocytes, using mutant mice bearing an astrocyte-specific deletion of Myd88 gene expression, would affect the obesity phenotype and leptin resistance induced by HFD consumption.

Animals
Animals were fed a standard diet (STD, Feedlab, Gyeonggi-Do, Korea) or HFD (D12492, Research Diets, New Brunswick, NJ, USA) ad libitum and given free access to tap water. All animals were maintained in temperatureand humidity-controlled rooms with a 12 h/12 h lightdark cycle, with the lights on from 7:00 a.m. to 7:00 p.m. Myd88 floxed (Myd88 fl/fl ) mice (stock no. 008888), glial fibrillary acidic protein (Gfap)-CreER T2 mice (Stock No. 012849), and Ai14 reporter mice (stock no. 007914) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Myd88 fl/fl mice were crossbred with Gfap-CreER T2 mice to generate Myd88 conditional knockout (KO) mice missing Myd88 specifically in cells expressing GFAP (Myd88 ΔGFAP ). Ai14 reporter mice were crossbred with Gfap-CreER T2 mice to label GFAP-positive astrocytes with tomato signals. Because the Gfap-CreER T2 mice expressed Cre recombinase under the control of the tamoxifeninducible GFAP promoter, 6-week-old Myd88 ΔGFAP mice and their littermate control (Myd88 fl/fl ) mice received daily injections for 5 days of tamoxifen (100 mg/kg, T5648, Sigma-Aldrich, St. Louis, MO, USA) dissolved in corn oil (C8267, Sigma-Aldrich). All animals and procedures used were in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee at the University of Ulsan (permission numbers: BJL-15-010, BJL-18-010, and BJL-19-010).

Ribo-Tag system
To analyze mRNA species that are specifically translated in hypothalamic astrocytes, we used the Ribo-Tag translational profiling system [22,23]. In this study, we used Rpl22 HA mice (Stock No. 011029, Jackson Laboratory), which have a loxP-flanked wild-type exon 4 followed by an identical exon 4 tagged with hemagglutinin (HA), as the Ribo-Tag animal. Crossbreeding Ribo-Tag mice with mice expressing Cre recombinase resulted in the deletion and replacement of the floxed wild-type exon 4 with the HA-tagged exon 4 in cells expressing Cre. The astrocyte-specific Myd88 KO (Myd88 ΔGFAP ) mice were crossbred with Rpl22 HA mice to generate Myd88 ΔGFAP ; Rpl22 HA mice that had both the HA-tagged ribosomal protein Rpl22 and the deletion of Myd88 in astrocytes. The Rpl22 HA mice were also crossed with control Myd88 +/+ -Gfap-CreER T2 (Gfap-Cre) mice, which resulted in control mice bearing an astrocyte-specific Ribo-Tag system (Gfap-Cre;Rpl22 HA mice).
RNA isolation with the Ribo-Tag system was conducted as previously described [22,23]. Briefly, dissected hypothalamus samples were collected from animals and homogenized. RNA was extracted from 10% of the cleared lysate and used as input. The remaining lysate was incubated with mouse anti-HA antibody for 4 h at 4°C followed by the addition of protein G agarose beads (LGP-1018B, Lugen, Gyeonggi-Do, Korea) and overnight incubation at 4°C. The beads were washed three times in high-salt solution. The bound ribosomes and RNA were separated from the beads with 30 s of vortexing, and RNA was further purified using a QIAGEN RNeasy Micro Kit (74034, Qiagen, Hilden, Germany). After RNA isolation, we obtained 10-20 ng of RNA sample/ hypothalamus. The RNA samples were then subjected to real-time PCR analysis.

Measurement of food intake and leptin administration
Five days before we began the food intake measurements, we moved the mice into individual cages and allowed them to acclimatize to their new environment. Food intake was measured for a week at 23-24 weeks of age during HFD feeding and calculated as an average daily food intake (Fig. 2a).
Body weight was measured every week during HFD feeding. To determine how leptin affected feeding behavior, mice were intraperitoneally (ip) injected with vehicle (saline) or recombinant mouse leptin (2 mg/kg; R&D Systems, Minneapolis, MN, USA) after overnight fasting. The food intake of the individually caged animals was monitored for 24 h after the injection, and then their body weights were measured.

Cannulation and administration of palmitic acid
For intracerebroventricular (icv) cannula implantation, mice were anesthetized by ip injection of tribromoethanol (250 mg/kg, Sigma-Aldrich) and placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA). The cannula (26 gage) was implanted into the right lateral ventricle (1.0 mm lateral, 0.3 mm posterior, and 2.4 mm ventral to the bregma) according to the Stereotaxic Mouse Brain Atlas (Paxinos G and Franklin KBJ, 2001, Academic Press, San Diego, CA, USA) and secured to the skull with dental cement. After 7 days of recovery, the mice were injected with vehicle [5% bovine serum albumin (BSA)] and palmitic acid (50 pmol/2 μl, Sigma-Aldrich) dissolved in BSA solution. Mice were sacrificed 1 h after the injection of palmitic acid.

Administration of 5-bromodeoxyuridine
For the analysis of Iba1-positive cell proliferation upon HFD, mice received daily ip injections with 5bromodeoxyuridine (BrdU, 100 mg/kg, Sigma-Aldrich) dissolved in saline for 5 days after HFD feeding for 8 weeks. On the 5th day of injection, mice were sacrificed 1 h after BrdU injection, and their brain sections were analyzed with immunohistochemistry.

IHC image analyses
The number of immuno-positive cells in the hypothalamic ARC was counted by an unbiased observer. The intensity of immuno-positive cells was measured using the ImageJ V 1.50 software (National Institutes of Health, Bethesda, MD). Region of interest (ROI) within an image was manually selected with the Mouse Brain Atlas for ARC or PVN (ARC: between − 1.46 and − 1.82 mm from bregma, PVN: between − 0.82 and − 1.06 mm from bregma). The images were converted to 8-bit images and threshold was applied. The images were binarized to separate the immuno-positive cells from the background. The fiber intensity and particle number of immuno-positive α-melanocyte-stimulating hormone (α-MSH) signals in the PVN were measured using the ImageJ software. The size of Iba1-positive cells in the ARC was measured using a thresholding parameter on the ImageJ software as a previous report [24].

Blood glucose measurement
Blood glucose was measured with a glucometer (One Touch Ultra, LifeScan, Milpitas, CA, USA). For glucose tolerance tests (GTTs), mice were given an ip injection of D-glucose (1 g/kg) after overnight fasting. For insulin tolerance tests (ITTs), mice were fasted for 4 h before ip injection of human insulin (0.75 IU/kg). Blood glucose levels were determined from the tail vein at 0, 15, 30, 60, and 120 min after injection.
Measurement of O 2 consumption, CO 2 production, and energy expenditure Metabolic parameters, O 2 consumption (VO 2 ), CO 2 production (VCO 2 ), and energy expenditure, of MyD88 fl/fl and MyD88 ΔGFAP mice were analyzed using an indirect calorimetry system (Promethion, Sable Systems, Las Vegas, NV, USA). VO 2 and VCO 2 were measured at 10 min intervals for each mouse. Mice were acclimated in the chambers for 48 h prior to data collection. The average values during the light and dark periods were calculated. Data acquisition and instrument control were coordinated by the MetaScreen software (version 2.3.12), and the obtained raw data were processed using Expe-Data (version 1.9.14, Sable Systems).

Real-time PCR
RNA was isolated from hypothalami using Trizol reagent (Sigma-Aldrich) or immunoprecipitation with HA antibody, as explained above, and reverse transcribed with MMLV reverse transcriptase (Beams Biotechnology, Gyeonggi-do, Korea). Gene expression was measured by real-time PCR using Evagreen qPCR Mastermix  -3′; L19 antisense primer, 5′-TTC AGC TTG  TGG ATG TGC TC-3′. Real-time PCR was performed using the StepOnePlus TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) for~40 cycles. Relative mRNA expression was normalized with the βactin or L19 mRNA level and calculated using the 2 −ΔΔCT method [25].

Statistical analyses
Statistical analyses were performed in the GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). All data are expressed as the mean ± SEM. The statistical significance between two groups was analyzed by unpaired Student's t test. Two-way analysis of variance (ANOVA) analyses followed by Bonferroni post hoc testing was performed to detect the significance of differences between two genotypes.

Results
Myd88 gene expression in astrocytes was increased by long-term HFD feeding To validate that long-term exposure to HFD caused reactive gliosis in the hypothalamus, mice were fed an HFD for 16 weeks, and IHC using antibodies against GFAP, a molecular marker for astrocytes, and Iba1, a marker for microglia, was performed with brain sections containing hypothalamic ARC (Fig. 1a, d). Consistent with previous reports [11,12,26], HFD feeding increased the number and intensity of GFAP-positive cells (Fig. 1b, c) and Iba1-positive cells (Fig. 1e, f).
To explore the function of MyD88 in hypothalamic astrocytes, we first used a Ribo-Tag system of transgenic (Gfap-Cre;Rpl22 HA ) mice that expressed HA-tagged ribosomal protein Rpl22 in astrocytes to identify the Myd88 mRNA species translated specifically in hypothalamic astrocytes. The IHC analysis identified specific HA signals in the GFAP-positive astrocytes ( Fig. 1 g). Real-time PCR using the Ribo-Tag system further revealed that HA-mediated immunoprecipitation occurred in the cells expressing GFAP, but not in those producing NeuN or Iba1 (Fig. 1h). The amount of Myd88 mRNA translated in hypothalamic astrocytes was increased by HFD feeding (Fig. 1i), suggesting that astrocyte MyD88 could play a role in the response to over-nutrition.
Astrocyte-specific Myd88 knockout alleviated HFDinduced hypothalamic gliosis It has been well established that coupling between TLRs and MyD88 initiates innate immunity in several types of peripheral cells and is also involved in the hypothalamic inflammatory responses linked to metabolic disorders [17,18,27]. To determine the effect of astrocyte MyD88 on HFD-induced astrogliosis and inflammation in the hypothalamus, we generated tamoxifen-inducible Myd88 gene KO specifically in GFAP-positive cells (Myd88 ΔG-FAP ). More than 83% of Myd88 mRNA expression was eliminated in the hypothalamic astrocytes of Myd88 ΔG-FAP ;Rpl22 HA mice, compared with that in the hypothalamic astrocytes of control Gfap-Cre;Rpl22 HA mice, as shown by a real-time PCR analysis of Ribo-Tag-purified mRNA (Fig. 2b).
Next, we measured the effect of this conditional Myd88 KO on HFD-induced astrogliosis in the hypothalamic ARC by counting GFAP-immuno-positive signals (Fig. 2c). The HFD-induced increase in the number and intensity of GFAP-positive cells was completely offset by Myd88 KO in astrocytes (Fig. 2d, e), suggesting the importance of MyD88 signaling in HFD-induced astrogliosis. The astrocyte-specific Myd88 KO also inhibited the HFD-induced increase in the number, intensity, and size of Iba1-positive cells in the hypothalamic ARC ( Fig. 2f-i). Furthermore, the Myd88 KO resulted in a decreased number of HFD-induced Iba1-positive cells that were incorporated with BrdU (Fig. 2j, k). These results suggested that astrocyte MyD88 signaling also participates in HFD-induced microglial proliferation and activation. Consistent with the decrease in HFD-induced astrogliosis in the mutant animals, astrocyte-specific Myd88 KO decreased the HFD-induced mRNA expression of Gfap, Iba1, and proinflammatory cytokines in the hypothalamus (Fig. 2l). Real-time PCR analyses of hypothalamic mRNA purified with the Ribo-Tag system showed that astrocyte- . a-f Immunohistochemical analyses were performed to determine changes in astrocytes and microglia in the hypothalamic arcuate nucleus (ARC) caused by HFD feeding. Representative images (a, d) and calculated data (b, c, e, f) indicate that GFAP-positive astrocytes (a-c) and Iba1-positive microglia (d-f) were increased by an HFD compared with a standard diet (STD) for 16 weeks (n=6-12 sections of 3-6 mice/group). g-i Ribo-Tag analyses of Myd88 mRNA expression in the hypothalamic astrocytes of transgenic mice (Gfap-Cre;Rpl22 HA ) expressing hemagglutinin (HA)-tagged ribosomal protein Rpl22 in GFAP-positive cells. g Representative images showing co-expression of HA and astrocyte-specific tomato signals in the hypothalamic ARC of Gfap-Cre;Rpl22 HA mice. h Real-time PCR data showing enrichment of Gfap mRNA (but not NeuN and Iba1 mRNA) in the RNA samples immunoprecipitated with HA antibody compared with the input RNA samples from hypothalamic extracts. i Ribo-Tag analyses showing that Myd88 mRNA expression in the hypothalamic astrocytes of Gfap-Cre;Rpl22 HA mice were increased by HFD feeding, compared with STD feeding, for 16 weeks (n=3-4/group). Data are presented as mean ± SEM. **p<0.01 and ***p<0.001. Scale bar = 100 μm The expression of Myd88 mRNA was determined using a real-time PCR analysis with RNA samples immunoprecipitated with HA antibody from hypothalamic extracts of Myd88 △GFAP ;Rpl22 HA mice that were generated by crossbreeding astrocyte-specific Myd88 KO mice (Myd88 △GFAP ) with transgenic mice (Rpl22 HA ) expressing HA-tagged Rpl22. Gfap-Cre;Rpl22 HA mice were used as the control (n = 4-5/group). c-i Control (Myd88 fl/fl ) mice and Myd88 △GFAP mice were fed a STD or HFD for 16 weeks, and their astrocytes and microglia in the hypothalamic ARC were analyzed with immunohistochemistry using GFAP and Iba1 antibodies. Representative images (c, f) and calculated data (d, e, g, h, i) show the effect of astrocyte-specific Myd88 KO (Myd88 △GFAP ) on the HFD-induced increase in the number and intensity of GFAP-positive cells (c-e) and Iba1-positive cells (f-h), and the size of Iba1-positive cells (i) in the ARC compared with the control Myd88 fl/fl mice (n=3-6 sections of 3-6 mice/group). j, k Representative images (j) and calculated data (k) indicate HFD-induced BrdU and Iba1 double-positive cells in the ARC of Myd88 △GFAP mice and control Myd88 fl/fl mice after HFD feeding for 8 weeks. White arrows indicate cells double positive for Iba1 and BrdU (n = 6 sections of 3 mice/group). l Real-time PCR analysis of RNA samples shows the expression of Gfap, Iba1, Tnf-α, Il-1β, Il-6, and Il-10 in the hypothalamus of Myd88 △GFAP mice and control Myd88 fl/fl mice after HFD feeding for 16 weeks (n = 3/group). m Hypothalamic expression of Tnf-α, Il-1β, Il-6, and Il-10 was determined using a real-time PCR analysis of RNA samples (immunoprecipitated with HA antibody) from hypothalamic extracts of Myd88 △GFAP ;Rpl22 HA mice and control Gfap-Cre;Rpl22 HA mice after HFD feeding for 16 weeks (n = 3-4/ group). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. ns, not significant. Scale bar = 100 μm (20 μm for higher magnification view in insets) specific Myd88 KO decreased HFD-induced expression of proinflammatory cytokines Tnf-α and Il-1β, but did not significantly affect the HFD-induced anti-inflammatory cytokines in the hypothalamic astrocytes (Fig. 2m).
Astrocyte-specific Myd88 KO affected hypothalamic gliosis triggered by palmitic acid Because elevated levels of circulating sFFAs can cause hypothalamic reactive gliosis that is accompanied by inflammatory responses during over-nutrition [26,28,29], we next determined the effect of astrocyte-specific Myd88 KO on the hypothalamic astrogliosis induced by the administration of palmitic acid, an sFFA. Palmitic acid induced an increase in the number and intensity of GFAP-positive astrocytes in the hypothalamic ARC, which was attenuated by Myd88 KO in the astrocytes ( Fig. 3a-c). Interestingly, astrocyte-specific Myd88 KO also caused a similar effect on palmitic acid-induced increase in the number and intensity of Iba1-positive microglia (Fig. 3d-f). Taken together, the current findings suggest that MyD88 signaling is a crucial molecular mediator of the hypothalamic gliosis induced by an elevation in circulating sFFAs.
Astrocyte-specific Myd88 KO did not affect metabolic phenotypes in the normal diet condition Before we determined whether HFD-induced metabolic disorder could be ameliorated by specific Myd88 KO in the astrocytes, we investigated the effect of astrocytespecific Myd88 KO on energy metabolism under the STD feeding condition. We examined alterations in metabolic parameters (food intake, body weight, and Data are presented as mean ± SEM. *p < 0.05 and ***p < 0.001. ns, not significant. Scale bar = 100 μm glucose metabolism) between the conditional KO (Myd88 ΔGFAP ) mice and control mice. The Myd88 ΔGFAP mice did not show any difference in food intake or body weight compared with control mice (Fig. 4a, b). Furthermore, the Myd88 ΔGFAP mice displayed normal glucose metabolism in the GTTs and ITTs (Fig. 4c-f). These results indicate that astrocyte-specific Myd88 KO did not cause metabolic abnormalities in the STD condition.

Astrocyte-specific ablation of Myd88 alleviated HFDinduced metabolic aggravation
To identify the pathological relevance of the reduced hypothalamic gliosis seen in the HFD-treated Myd88 ΔGFAP mice, both control and Myd88 ΔGFAP mice were fed an HFD for 16 weeks, and then their metabolic parameters were measured. The Myd88 ΔGFAP mice fed an HFD revealed a significant decrease in food intake and calorie intake compared with the HFD-fed control mice (Fig. 5a, b). Additionally, HFD-induced weight gain was significantly alleviated in the Myd88 ΔGFAP mice compared with control mice during the observation period (Fig. 5c). Along with the difference in body weight gain during HFD feeding, the peripheral metabolic organs, such as the liver and perirenal fat, of the Myd88 ΔGFAP mice weighed less than those of the control mice (Fig. 5d). Accordingly, the Myd88 ΔGFAP mice displayed improved glucose metabolism, as shown in GTTs and ITTs, after long-term exposure to HFD (Fig. 5e-h). To further investigate the effect of astrocyte-specific Myd88 KO on energy expenditure, we measured multiple metabolic parameters using indirect calorimetry. The Myd88 ΔGFAP mice showed significant elevations of VO 2 , VCO 2 , and energy expenditure after long-term exposure to HFD, compared with control mice (Fig. 5i-m). Collectively, these observations demonstrate that selective ablation of the Myd88 gene in astrocytes ameliorates diet-induced obesity (DIO) and impaired glucose metabolism by affecting food intake and energy expenditure.
Astrocyte-specific deletion of the Myd88 gene ameliorated long-term HFD feeding-induced leptin resistance It has been well established that hypothalamic inflammation is a primary cause of leptin resistance, which is deeply associated with obesity pathogenesis. Therefore, we next investigated the responsiveness of the Myd88 ΔG-FAP mice to leptin after 16 weeks of HFD feeding. An ip administration of leptin (2 mg/kg body weight) effectively reduced food intake and body weight in both control and Myd88 ΔGFAP mice fed a STD diet, indicating that astrocyte-specific Myd88 KO did not affect leptin  responsiveness under non-obesity conditions. Leptininduced reduction in food intake and body weight disappeared completely in control (Myd88 fl/fl ) mice fed an HFD for 16 weeks, indicating a condition of leptin resistance. However, Myd88 ΔGFAP mice fed an HFD for 16 weeks continued to display a decrease in food intake and body weight in response to leptin, indicating ameliorated leptin resistance (Fig. 6a, b). Because phosphorylation of signal transducer and activator of transcription 3 (STAT3) is a cellular event that reflects the activation of leptin signaling, we determined leptin-induced STAT3 phosphorylation (pSTAT3) in the Myd88 ΔGFAP mice. The Myd88 ΔGFAP mice preserved leptin-induced pSTAT3 in the hypothalamic ARC after 16 weeks of HFD feeding, whereas the control mice lost the normalcy of leptin-induced pSTAT3 after long-term HFD feeding (Fig. 6c, d). Furthermore, leptin induced an increase in the number and intensity of α-MSH fibers in the paraventricular nucleus (PVN) of the Myd88 ΔGFAP mice after HFD feeding for 16 weeks, whereas it failed to induce a significant change in the α-MSH fibers of the PVN of the control Myd88 fl/fl mice ( Fig. 6e-g). Collectively, these observations suggest that astrocyte MyD88 signaling is closely correlated with leptin responsiveness during long-term over-nutrition.

Discussion
In the present study, we found that Myd88 expression in hypothalamic astrocytes was increased by long-term HFD feeding and that astrocyte-specific ablation of Myd88 ameliorated the obesity-related metabolic phenotype induced by HFD consumption. The current observations demonstrate that MyD88 signaling in astrocytes is a critical contributor to the hypothalamic inflammation-induced pathogenesis of obesity.
In this study, we focused on the role of MyD88 signaling in astrocytes as a mediator of hypothalamic inflammation during HFD feeding. Interestingly, we found that HFDinduced reactive gliosis was decreased in hypothalamic ARC of mutant mice lacking Myd88 expression in astrocytes compared to control mice. Moreover, a direct icv infusion of sFFAs induced reactive gliosis in hypothalamic ARC of control mice, but not in astrocyte-specific Myd88 KO mice. These results suggested that MyD88 signaling in astrocytes plays a critical role in the hypothalamic reactive gliosis induced by HFD and sFFAs. In animals chronically fed an HFD, reactive astrocytes in the hypothalamus induced neuronal damage by secreting proinflammatory cytokines [14,26]. In this DIO model, the proinflammatory cytokines released by hypothalamic astrocytes and microglia activated cytokine receptors on the hypothalamic proopiomelanocortin (POMC) and agoutirelated peptide (AgRP) neurons [12,20,30,31]. In the current study, astrocyte-specific Myd88 KO resulted in a decrease in activation of microglia in the ARC and expression of proinflammatory cytokines in the hypothalamus during HFD feeding. These changes might be due to that astrocyte-specific deletion of Myd88 gene caused a decrease in HFD-induced intracellular signaling for the activation of proinflammatory cytokine expression in the hypothalamic astrocytes. Since the ARC of the hypothalamus is considered as a major site for the actions of POMC and AgRP neurons that dynamically participate in the central control of energy homeostasis, we focused on the action of astrocytes in the hypothalamic ARC. However, we could not exclude a possible contribution of astrocytes in other sites of the hypothalamus including the median eminence and third ventricular linings to the development of inflammatory processes. Therefore, further studies are required to identify subpopulations of hypothalamic astrocytes that are important in the initiation of hypothalamic inflammation during a long-term exposure to over-nutrition.
HFD-induced hypothalamic reactive gliosis and inflammatory responses are important factors in leptin sensitivity during HFD consumption [14,20]. Despite high circulating leptin levels, over-nutrition-induced obese mice show a reduced responsiveness to the appetite-and weight gain-suppressing effects of leptin, which is generally called leptin resistance and is a critical element in the development of obesity. It has been proposed that development of leptin resistance is coupled to multiple cellular events including abnormalities of leptin signaling, leptin transportation, and leptin receptor trafficking [32]. Particularly, it is currently accepted that the hypothalamic gliosis accompanied by enhanced inflammation responses is regarded as a crucial pathological element during the over-nutrition period [12,14,20]. In line with these notions, we (See figure on previous page.) Fig. 5 Astrocyte-specific Myd88 KO affects HFD-induced obesity. Astrocyte-specific Myd88 KO (Myd88 △GFAP ) mice and control Myd88 fl/fl mice were fed an HFD for 16 weeks, and then their metabolic parameters were measured. a, b Average daily food intake (a) and average daily calorie intake (b) were measured for a week at 23-24 weeks of age during HFD feeding (n = 4/group). c The accumulated weight gain of the mice was observed every week during HFD feeding (n = 4/group). HFD, high-fat diet. d The weight of adipose tissues was measured at 24 weeks of age (n = 4/group). BAT, brown adipose tissue. e-h Glucose tolerance testing (e, f) and insulin tolerance testing (g, h) were carried out in mice after HFD feeding for 16 weeks (n = 5-9/ group). AUC, area under curve. i-m Indirect calorimetry measurements were performed in metabolic cages to determine changes in the oxygen consumption (VO 2 ) (i, j), carbon dioxide generation (VCO 2 ) (k, l), and energy expenditure (m) of mice after HFD feeding for 16 weeks (n = 8/group). Data are presented as mean ± SEM. *p < 0.05, and **p < 0.01 observed that the Myd88 ΔGFAP mice displayed antiobesity phenotype in association with improved responsiveness to leptin after long-term HFD feeding. In accordance with the behavioral observations, we confirmed the leptin-triggered induction of pSTAT3, a general molecular marker for the leptin responsiveness, in the hypothalamic ARC. Given that leptin signaling operates the activity of POMC neurons that govern hypothalamic cells expressing melanocortin receptors by releasing α-MSH, we further validated improved responsiveness of the Myd88 KO mice to leptin by identifying the enhanced innervation of α-MSH in the hypothalamic PVN, even after long-term HFD feeding. These observations strengthen the underappreciated role of astrocytic TLR-MyD88 signaling in the regulation of over-nutrition-induced hypothalamic inflammation and Fig. 6 Astrocyte-specific Myd88 KO enhances leptin responsiveness after long-term HFD feeding. a, b To identify the effects of astrocyte-specific Myd88 KO (Myd88 △GFAP ) on HFD-induced leptin resistance, mice were fed an HFD for 16 weeks, and their food intake (a) and body weight (b) were measured for 24 h after an intraperitoneal injection of leptin (2 mg/kg) or vehicle (n = 3-4/group). c, d Representative immunohistochemical images (c) and calculated graphs (d) show that leptin-induced pSTAT3 in the arcuate nucleus had deteriorated in the control Myd88 fl/fl mice after 16 weeks of HFD feeding. However, the Myd88 △GFAP mice fed the HFD for 16 weeks showed a leptin-induced pSTAT3 level similar to those with STD feeding. e-g Representative images (e) and calculated data (f, g) reveal that leptin induced an increase in α-MSH immuno-positive signals in the paraventricular nucleus of the Myd88 △GFAP mice but not the control Myd88 fl/fl mice after 16 weeks of HFD feeding (n = 3 sections of 3 mice/ group). Data are presented as mean ± SEM. *p < 0.05, **p<0.01 and ***p < 0.001. ns, not significant. Scale bar = 100 μm metabolic abnormalities. Notably, previous literatures have suggested that leptin receptors are present in hypothalamic astrocytes and participate in the central control of energy metabolism [33][34][35]. However, active role of the leptin receptors in hypothalamic astrocytes is still controversial. Thus, this study raised an important question whether MyD88 is connected to the leptin receptor signaling in hypothalamic astrocytes. It is well-known that overnutrition-induced inflammatory factors activate NF-κB signaling, which upregulates negative regulators of leptin signaling, such as suppressor of cytokine signaling 3, and thus inhibits pSTAT3 signaling [20,36,37]. In addition, elevated activity of TLRs and MyD88 coupling leads to the activation of NF-κB signaling in hypothalamic neurons [20,21]. Therefore, these evidences and current findings together suggest that MyD88 signaling in astrocytes is important in leptin resistance caused by HFD-induced inflammation and obesity pathogenesis.
Although studies to understand the mechanisms that underlie leptin resistance during HFD consumption have focused on the role of astrocytes and microglia, the cooperative actions and relationships between them during HFD-induced neuroinflammation remain largely unknown. Several reports have revealed that crosstalk occurs between astrocytes and microglia through the release of signaling factors (such as cytokines and chemokines) that contribute to the pathogenesis of neuroinflammation and neurodegeneration [31,38]. A recent in vitro study showed that the accumulation of lipid droplets in hypothalamic astrocytes could be induced by sFFA treatment and led to the activation of microglia through inflammatory cytokines [39]. In our study, astrocyte-specific Myd88 KO blocked HFDinduced microglial activation. In line with previous reports, this result might indicate that astrocyte-specific Myd88 KO decreased HFD-induced expression of cytokines in astrocytes, which further indicates crosstalk between astrocytes and microglia and the importance of astrocyte MyD88 signaling in HFD-induced obesity.

Conclusions
The present study reports that MyD88 signaling in astrocytes is a key mediator of obesity pathogenesis and highlights its contribution to over-nutrition-induced reactive gliosis and leptin resistance in the hypothalamus. These observations suggest that MyD88 signaling in hypothalamic astrocytes could be an important novel target for the treatment of metabolic disorders such as leptin resistance and obesity.